Transient DNA hypomethylation is observed when murine erythroleukemia cells (MELC) are induced to differentiate. The fact that this phenomenon takes place in the absence of DNA replication led us to the discovery of a novel reaction whereby a modification in the pattern of DNA methylation is brought about by the specific removal of methyl cytidine (mC) residues and their replacements by cytidine. The transient nature of DNA hypomethylation indicates that this event must be followed by de novo DNA methylation, and indeed we have previously shown that such is the case. Treatment of MEL cells with 3-DZA ( 3-deaza-Ado) and homocysteine will completely inhibit the expression of the differentiated state (measured at 72-96 hours) if these agents are added between 8 and 20 hours after induction; if 3-DZA + Hcy are added together with the inducer but removed 8 hrs later there is no effect on differentiation. If administration of DZA + Hcy is delayed for 24 hrs after induction differentiation proceeds normally. We have established furthermore that the reaction catalyzed by 5-mC replacase is inhibited in parallel fashion and with the same time frame. Thus during the process of differentiation, but long before its expression, there is "window" when one or more biochemical reactions are susceptible to inhibition by DZAHcy. Since we have recently observed that HMBA induced hypomethylation is not affected by inhibition of protein synthesis we have reached the tentative conclusion that 5-mC replacase is present in an inactive form in exponentially growing MEL cells and becomes active upon induction through carboxymethylation. The striking correspondence in the timing of the inhibition of differentiation and of the MMBA induced DNA hypomethylation produced by 3-DZA + Hcy adds weight to the hypothesis that this limited and specific modification of DNA structure is one in a series of events that are required for the expression of the differentiated genotype.